Our long term goal is to develop genetic approaches that eventually could be used for the treatment of Mitochondrial Disorders associated with mitochondrial DNA (mtDNA) mutations. Although some of the approaches tested by us and others in the past showed some promise, they are inefficient and limited to correcting problems in a few genes. We now propose to develop approaches that are more efficient in improving oxidative phosphorylation in cells harboring heteroplasmic mtDNA mutations (i.e. a mixture of wild-type and mutated mtDNA). The approach is based on the "mtDNA heteroplasmy shift" concept, where, by genetic manipulation, levels of the wild-type genome are increased in relation to the mutated genome. Our proposal will apply two related but different tools to induce mtDNA heteroplasmy shift. The first one is the use mitochondrially-targeted restriction endonucleases that can specifically cleave mitochondrial genomes containing mutated sites. We have shown that such approach can shift mtDNA heteroplasmy efficiently in cultured cells. We now propose to continue these studies in cultured cells and to test the efficiency and safety of the procedure in transgenic mice. Because restriction endonucleases are also limited by the number of recognition sites, we propose to expand this approach by using a second tool, namely mitochondrially-targeted chimeric Zinc Finger Proteins and type IIS restriction endonucleases' catalytic domain. Zinc Finger Proteins can be designed and fined tuned for the recognition of novel DNA sites. Once the DNA binding specificity is obtained, the addition of the catalytic domain from type IIS restriction endonucleases (Fokl) to the Zinc Finger DNA binding region would create molecules with a potential to shift mtDNA heteroplasmy of mutations that are prevalent in the patient population.